This invention concerns peptides and pharmaceutical compositions that are useful to inhibit formation of protease resistant prion proteins (PrPres), such as the protease resistant prion proteins associated with transmissible spongiform encephalopathies.
Transmissible spongiform encephalopathies (TSE) are fatal neurodegenerative diseases that include such human disorders as sporadic and familial Creutzfeldt-Jakob disease (CJD), kuru, fatal familial insomnia, and Gerstmann-Straussler-Scheinker syndrome. Animal forms of the diseases include scrapie in sheep and bovine spongiform encephalopathy in cattle. These diseases are characterized by the formation and accumulation in the brain of an abnormal proteinase K resistant isoforn (PrPres) of a normal protease-sensitive host-encoded prion protein (PrPsen). PrPres is formed from PrPsen by a post-translational process involving conformational changes that convert the PrPsen into a PrPres molecular aggregate having a higher xcex2-sheet content. The formation of these macromolecular aggregates of PrPres is closely associated with TSE-mediated brain pathology in which amyloid deposits of PrPres are formed in the brain, which eventually becomes xe2x80x9cspongiformxe2x80x9d (filled with holes).
In the past, the TSE diseases were a medical curiosity because the transmissible agent was difficult to inactivate with heat, radiation or chemicals that would be expected to inactivate infectious living organisms such as bacteria and viruses. Instead, this class of diseases appeared to be transmitted by exposure to an unusual agent, for example by ritual cannibalism in the Foret people of New Guinea, or feeding of animal parts to cattle in bovine spongiform encephalopathy (BSE). Iatrogenic CJD has also been caused by administration of human growth hormone derived from cadaveric pituitaries, transplanted dura mater and corneal grafts, as well as exposure of surgeons to affected tissue during neurological procedures. The TSE diseases took on new urgency, however, when it appeared that cross-species infection of humans in Europe may have occurred, perhaps from the ingestion of beef from affected cows. That development has further stimulated an international search for a better understanding of the pathophysiological mechanism of the disease, and possible treatments.
The presence of a native prion protein (PrP) has been shown to be essential to pathogenesis of TSE. The cellular protein PrPsen is a sialoglycoprotein encoded by a gene that in humans is located on chromosome 20. The PrP gene is expressed in neural and non-neural tissues, with the highest concentration of its mRNA being in neurons. The translation product of the PrP gene consists of 253 amino acids in humans, 254 in hamsters and mice, 264 amino acids in cows, and 256 amino acids in sheep (all of these sequences are disclosed in U.S. Pat. No. 5,565,186, which describes methods of making transgenic mice that express species specific PrP. Other sequence information is included in Locht, C. et al., Proc. Natl. Acad. Sci. USA 83:6372-6376, 1986; Kretzschmar, H. A. et al., DNA 5:315-324, 1986; Yoshimoto, J. et al., Virus Genes 6:343-356, 1992; Goldmann, W. et al. Proc. Natl. Acad Sci. USA 87:2476-2480, 1990). In prion protein related encephalopathies, the cellular PrPsen is converted into the altered PrPres that is distinguishable from PrPsen in that PrPres (1) aggregates; (2) is proteinase K resistant in that only approximately the N-terminal 67 amino acids are removed by proteinase K digestion under conditions in which PrPsen is completely degraded; and (3) has an alteration in protein conformation in which the amount of xcex1-helical conformation for PrPsen is reduced, and the amount of xcex2-sheet conformation for PrPres is increased.
If PrPsen is not expressed in the brain tissue of animal recipients of scrapie-infected neurografts, no pathology occurs outside the graft, demonstrating that PrPres and PrPsen are both required for the pathology (Brander et al., Nature 379:339-343, 1996). The long latency period between infection and the appearance of disease (months to decades depending on species) has prompted the development of a cell-free in vitro test, in which PrPres induces the conversion of PrPsen to PrPres (Kocisko et al, Nature 370:471-474, 1994). See also Prusiner et al., WO 97/16728 published May 9, 1997. The in vitro interaction between PrPres and PrPsen occurs with species and strain specificities that mimic TSE species barrier effects and strain differences in vivo (Kocisko et al., Proc Natl Acad Sci USA 92, 3923-3927, 1995; Bessen et al., Nature 375, 698-700, 1995; Bossers et al., Proc. Natl. Acad. Sci. USA 94, 4931-4936, 1997; Raymond et al., Nature 388, 285-288, 1997), hence in vitro cell free culture techniques are considered to accurately predict pathological developments in the brains of infected animals. These in vivo and in vitro observations indicate that direct interactions between PrPres and PrPsen form PrPres and promote TSE pathogenesis.
Small synthetic peptides containing certain PrP sequences have previously been shown to spontaneously aggregate to form fibrils with a high degree of xcex2-sheet secondary structure of the type seen in the insoluble deposits in TSE afflicted brains (Gasset et al. Proc. Natl. Acad. Sci. USA 89, 10940-10944, 1992; Come et al., Proc. Natl. Acad. Sci. USA 90, 5959-5963, 1993; Forloni et al., Nature 362, 543-546, 1993; Hope et al., Neurodegeneration 5, 1-11, 1996). Moreover, other synthetic PrP peptides have been shown to interact with PrPsen molecules to form an aggregated complex with increased protease-resistance (Kaneko et al., Proc. Natl. Acad. Sci. USA 92, 11160-11164, 1995; Kaneko et al., J. Mol. Biol. 270, 574-586, 1997). The PrP derived synthetic peptide Ala Gly Ala Ala Ala Ala Gly Ala (SEQ. ID. NO.1) from positions 113 to 120 of the PrP peptides has been described as the most highly amyloidogenic peptide in the protein (Gasset, M., et al., =Proc. Natl. Acad. Sci. USA 89, 10940-10944, 1992), which indicates that it would be expected to promote the formation of PrPres.
Holscher et al. (J. Virol. 72:1153-1159, 1998) recently showed that a mutant mouse PrP lacking the sequence from 114 to 121 (spanning the highly amyloidogenic region) is not converted into a proteinase K-resistant isoform after expression in scrapie-infected mouse neuroblastoma cells. This finding further supported the idea that this highly hydrophobic sequence promoted PrPres formation. U.S. Pat. No. 5,618,673 disclosed a scrapie specific palindromic oligonucleotide that hybridized to the DNA of scrapie infected tissue for use in diagnostic assays. U.S. Pat. No. 5,679,530 described a prion binding protein and peptide that were said to be useful in the non-histologic diagnosis of prion diseases.
Although these discoveries have supported the grim suggestion that certain PrP sequences can promote the formation of insoluble PrP deposits in the brain and elsewhere in the body, they have done little to provide an approach for inhibiting the formation of such deposits. However, U.S. Pat. No. 5,276,059 disclosed that mammalian diseases associated with amyloid protein formation, and the conversion of PrPsen to PrPres, could be treated by administering Congo Red dye to the mammal. U.S. Pat. No. 5,134,121 disclosed the use of a nerve growth blocking peptide to treat prion associated diseases.
Nonetheless, the need still remains for agents that will specifically inhibit the formation of PrPres, and by extension prevent or slow the deposition of amyloid deposits in the tissues of animals that have been exposed to a TSE, or are suffering from a neurodegenerative disorder having the characteristics of a spongiform encephalopathy.
The present invention takes advantage of the surprising finding that certain synthetic peptides, which incorporate the most amyloidogenic region Ala Gly Ala Ala Ala Ala Gly Ala (SEQ. ID. NO. 1) of the PrP protein, can actually inhibit the formation of PrPres under conditions where it would otherwise be formed. In one embodiment, the invention includes a purified peptide comprising or consisting essentially of XZ, where X includes at least two consecutive amino acid residues, such as the carboxy terminal residues of the dipeptide Gly Ala from SEQ. ID. NO. 1, and Z is a peptide region that, in cooperation with X, inhibits conversion of protease sensitive prion protein (PrPsen) to protease resistant prion protein (PrPres) under conditions in which PrPres would otherwise be formed. In particular embodiments, X may also be the tetrapeptide Ala Ala Gly Ala (SEQ. ID. NO. 2), the hexapeptide Ala Ala Ala Ala Gly Ala (SEQ. ID. NO. 3), or the octapeptide Ala Gly Ala Ala Ala Ala Gly Ala (SEQ. ID. NO. 1), and Z is Val Val Gly Gly Leu Gly Gly Tyr (SEQ. ID. NO. 4) or Val Val Gly Gly Leu Gly Gly Tyr Met Leu Gly Ser Ala Met Ser Arg Pro Met Met His Phe (SEQ. ID. NO. 5), Val Val Gly Gly Leu Gly Gly Tyr Met Leu Gly Ser Ala Met Ser Arg Pro Ile Ile His Phe (SEQ. ID. NO. 24) or a subsequence or variant thereof that interferes with the conversion of PrPsen to PrPres.
It has been found that even very short synthetic peptides, such as peptides consisting essentially of amino acid residues 119-128 (SEQ. ID. NO. 6), are able to inhibit the formation of PrPres. Hence the invention includes peptides XZ where X consists of at least the last two consecutive amino acids (Gly Ala) of SEQ. ID. NO. 1, and Z comprises at least the first eight amino acids of SEQ. ID. NO. 5. In more specific embodiments, X consists of at least the last four, six or eight consecutive amino acids of SEQ. ID. NO. 1, and Z consists of at least the first sixteen or even all twenty-one amino acids of SEQ. ID. NO. 5 or SEQ. ID. NO. 24. In a particularly disclosed embodiment, the purified peptide consists of SEQ. ID. NO. 8 (HaP109-141), which has an IC50 of about 30 xcexcM in inhibiting the cell free conversion of PrPsen to PrPres.
In yet other embodiments, the purified peptide consists of an amino acid sequence which has an amino terminal Gly Ala motif, or a variant with conservative substitutions therein; and has a carboxyterminal motif that is homologous to at least positions 120-128 of PrP (which is conserved across all known species), such that when exposed to PrPsen in therapeutically effective amounts, the peptide specifically inhibits formation of PrPres in the presence of PrPsen, under conditions where PrPsen would otherwise be formed. The specificity of the inhibition is sufficient, in such embodiments, that the IC50 of the inhibitory peptide is less than about 1000 xcexcM, for example less than about 550-600 xcexcM.
In other embodiments, the peptide comprises a subsequence of at least two amino acid residues from SEQ. ID. NO. 1, and a subsequence of SEQ. ID. NO. 13, but not a sequence of ten or more consecutive amino acid residues from positions 90-100 or 90-105 of HaPrP as shown in FIG. 10. In other embodiments, the peptide is a variant of the amino acid sequences discussed herein, but without amino acid substitutions that would be expected to change the conformation of the peptide. Substitutions that change the conformation of the synthetic peptide are avoided in these embodiments.
The invention also includes a pharmaceutical composition comprising a peptide, or a variant or mimetic thereof, and a pharmaceutically acceptable carrier or diluent. The pharmaceutical composition may be used as a diagnostic agent (for example to detect the presence of PrPres in a body fluid such as blood or cerebrospinal fluid), or may be in a unit dose form of a therapeutic agent for the treatment of spongiform encephalopathy. The invention also therefore includes a method of treating a spongiform encephalopathy, such as a TSE, by administering a therapeutically effective amount of the pharmaceutical composition to an animal that has been exposed to the transmissible agent, or which is exhibiting signs, symptoms or laboratory evidence of a TSE. If the animal is merely suspected of having been exposed to a TSE, the treatment is a prophylactic method of preventing the progression of the disease. In a situation where the animal is already believed to be exhibiting signs or symptoms of the disease, the treatment is also a method of improving the neurological or other biological condition of the animal.
The invention also includes in vitro methods for the inhibition of the conversion of PrPsen to PrPres, in which PrPsen is exposed to the inhibitory peptides in the presence of PrPres to inhibit the conversion of PrPsen to PrPres, and a method of screening for variants, analogs and mimetics of the inhibitory peptides that inhibit the conversion reaction in the assay. Also included are a specific binding agent (such as a monoclonal or polyclonal antibody) that binds an inhibitory peptide.
Also provided by the invention are nucleic acid molecules encoding the peptides disclosed herein, as well as vectors including these nucleic acid molecules.
The peptides of the present invention are particularly useful in designing analogs, derivatives or mimetics for use as a diagnostic agent or as a therapeutic inhibitor of the conversion of PrPsen to PrPres. The invention therefore also includes analogs, derivatives or mimetics of the disclosed peptides, as well as methods for screening such compounds which inhibit conversion of PrPsen to PrPres. The screening method includes contacting PrPsen with PrPres and a disclosed peptide, or peptides having one or more conservative amino acid substitutions, or analogs, derivatives or mimetics thereof, and determining whether the peptide, analog, derivative or mimetic inhibits conversion of PrPsen to PrPres.
Specific preferred embodiments of the present invention will become more evident from the following detailed description.